Neuropeptides play a key role in the regulation of a variety of physiological functions in insects. For example, they are involved in embryonic and post-embryonic development (e.g. molting, diapause and metamorphosis), in homeostasis, osmoregulation, diuresis and digestion. Insect neuropeptides are also known to control essential behavioral patterns such as migration, mating and oviposition.
Insect neuropeptides are involved in a variety of physiological processes, and therefore have great potential as pest control agents, based on the interference with their activity. The present invention addresses one step in the course of the neuroendocrine regulation, namely, binding of the neuropeptides to their receptors and activation of their target organ. Specifically, the present invention deals with the inhibitory activity of peptide fragments derived from the PK/PBAN family of peptides.
Sexual communication between male and female moths is regulated by sex pheromones. Sex pheromones are synthesized and secreted by the female from the pheromone gland. The inability of the female to produce sex pheromones results in a marked decrease in mating and thus, significantly reduces the insect population. Due to their important role in mating, sex pheromones have been studied intensively. For instance, it has been found that moths sex pheromones consist of blends of C10-C18 aliphatic compounds. The diversity between pheromones of different species is indicated by differences in the chain length, the position and configuration of the olefinic bonds, and by the chemical nature of the functional group. Most pheromones are aldehydes, alcohols or acetates. Some, however, may appear as epoxides, ketones and hydrocarbons.
It has been found that a neuroendocrine factor, termed PBAN, is involved in the regulation of sex pheromone biosynthesis. PBAN is a linear C-terminally amidated peptide containing 33 amino acids. PBAN's structure has been fully identified, and since 1989, its primary amino acid sequences has been revealed in over twenty moth species. PBAN is synthesized in the brain and subesophageal ganglion that projects to the corpora cardiaca, and in ganglia of the ventral nerve cord. The peptide is either transported to its target organ via the hemolymph or acts locally. It is present in both male and female moth and its biological activity is mediated by cAMP and depends on the presence of Ca−+ ions.
Further studies of insect neuropeptides revealed that the C-terminal sequence of PBAN—(X═S) FXPRLa (SEQ ID NO: 1)—is also present in other insect neuropeptides (where X represents S, T, G or V). All peptides sharing the above sequences were grouped into one family, which was designated as the FXPRLa family or the PK/PBAN family. In addition to the pheromonotropic activity of this family, PK/PBAN peptides and fragments derived therefrom, have also been shown to control cuticular melanization, muscle contraction and embryonic and pupal diapause.
JP 4-208300 describes the full 33 amino acid sequence of the PBAN peptide isolated from silkworms, as well as some of its fragments.
U.S. Pat. No. 5,032,576 relates to the isolation, characterization and synthesis of PBAN from Helicoverpa zea. U.S. Pat. No. 5,032,576 uses the full length PBAN and several analogs thereof (all of which are 33 amino acid long) for controlling female moths or larvae. However, U.S. Pat. No. 5,032,576 does not suggest or even hints towards the possibility of using shorter PBAN fragments.
One way to inhibit PK/PBAN peptides is by antagonists, which are selective inhibitors capable of blocking the receptor site of the neuropeptide, and thus, preventing from the endogenous peptide to bind to the receptor and exert their biological activity. Therefore, PK/PBAN antagonists can serve as specific inhibitors for their activity, and may serve as potential insecticides.
A substantial problem/challenge in the implementation of the above strategy for insect management is that there are no defined protocols to convert peptide agonists into antagonists. In addition, neuropeptides are unstable and readily attacked by enzymes. Therefore, developing neuropeptides as antagonistic compounds is not common. One way to overcome the problem is based on searching and creating peptidomimetic compounds which show enhanced biological stability. In order to design peptidomimetic antagonists, the structure-activity relationship of the peptides has to be revealed, conformationally constrained analogs based on their active site have to be designed and synthesized, then the active and inactive residues in those molecules have to be identified and peptidomimetic compounds containing the active residues have to be designed, synthesized and tested for stable antagonistic activity. This is usually a highly laborious and long process based on availability of many peptide libraries as well as a detailed instrumental chemical analysis combined with highly advanced in silico computational analysis. The entire operations requires expertise in peptide chemistry and instrumental analytical chemistry, without any promise or means to predict success. Once completed, such an approach leads to the discovery of many peptidomimetic compounds, most of which are very expensive in production and are also found, in many cases, to be ineffective.
Accordingly, one object of the invention is to simplify the process and to provide novel peptide-based antagonists which show high inhibitory potency and high stability and bioavailability on one hand, and which are simple to design and synthesize on the other. In addition, another object is to make said production cost-effective. Said antagonists are 5 to 6 amino acids long, and are derived from the PK/PBAN sequence.
In order to be effective, insecticides should be able to penetrate the insect's cuticle and/or be stable upon ingestion. Therefore, it is preferred to use insecticides with a low molecular weight. Accordingly, one embodiment of the invention are low molecular weight peptides which demonstrate high cuticle-penetration capabilities as well as stability upon ingestion. In another embodiment, the present invention relates to peptides which include a supplementary residue which further increases their cuticle- and oral penetration capabilities.